PROJECT SUMMARY/ABSTRACT Age and sex are the primary risk factors for developing Alzheimer?s disease (AD), with a higher incidence in women at all ages from 60-100 years old. Mechanisms underlying the cause of sex differences in AD are not well understood, preventing the development of sex-specific therapeutic interventions. Chronic, low-grade inflammation is characteristic of brain aging and neurodegeneration. As the sentinel macrophages of the CNS, microglia play an important role in sensing and responding to changes in the CNS milieu. Microglia monitor the CNS for foreign/?non-self? and endogenous substances, serving both neuroprotective and neurodegenerative functions. Under normal physiological conditions, microglia retain a relatively quiescent, surveillance phenotype; however stimulation can polarize microglia to the pro-inflammatory M1 phenotype or anti-inflammatory M2 phenotype. Control of microglial phenotypic switching involves regulation of DNA modifications, principally methylation and hydroxymethylation of cytosines, which control genome accessibility and gene expression. Exuberant activation of microglial-specific inflammatory pathways with age in females and sexual divergence in DNA modifications with age point to an epigenetic role in sex differences in brain aging and neuroinflammation. However, paired epigenomic and transcriptomic studies have not been conducted in hippocampal microglia with age between sexes. To address this barrier to progress we developed microglial-specific, tamoxifen-inducible, transgenic NuTRAP models to allow isolation of nucleic acids (DNA & RNA) from lineage-traced microglia. In Aim 1, microglial-specific hippocampal changes in mC/hmC with aging will be examined by whole genome oxidative bisulfite sequencing (WGoxBS). Epigenomic data, and the paired transcriptomic data from the same animals will be used to determine the role of altered modification patterns in age-related changes in gene expression, enrichment of differential modifications in regulatory regions of the genome, and to identify genomic loci for future epigenome editing experiments. Although sexual divergence is documented in some current rodent models of AD, an acknowledged limitation in the field is that transgenic animals develop AD neuropathology at a young age, unlike sporadic AD, which occurs late in life. New AD models like amyloid-beta (A?) oligomer infusion allow for examination of AD-related A? neuropathology in the context of brain aging and sex effects. In Aim 2, we combine A?-oligomer infusion with our novel NuTRAP model to selectively analyze microglial-specific epigenomic and transcriptomic changes in the context of A? neurotoxicity. Further, immunohistochemistry and cytokine/chemokine panels are used readouts for gliosis, to assess the effect of aging on A?-induced microglial reactivity between sexes. If successful, these studies will provide insights into sexually divergent microglial function with aging and A?-related neuropathology and identify targets for development of sex-specific therapeutic interventions. Future studies will also use these findings to examine the regulators of sexual divergence with brain aging.